Microcapsules having multiple shells and method for the preparation thereof

ABSTRACT

Single-core and multi-core microcapsules are provided, having multiple shells, at least one of which is formed of a complex coacervate of two components of shell materials. The complex coacervate may be the same or different for each shell. Also provided are methods for making the microcapsules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/423,363 filed Nov. 4, 2002, which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to microcapsules having multiple shells, tomethods of preparing microcapsules and to their use.

BACKGROUND OF THE INVENTION

Microcapsules are small particles of solids, or droplets of liquids,inside a thin coating of a shell material such as starch, gelatine,lipids, polysaccharides, wax or polyacrylic acids. They are used, forexample, to prepare liquids as free-flowing powders or compressedsolids, to separate reactive materials, to reduce toxicity, to protectagainst oxidation and/or to control the rate of release of a substancesuch as an enzyme, flavour, a nutrient, a drug, etc.

Ideally, a microcapsule would have good mechanical strength (e.g.resistance to rupture) and the microcapsule shell would provide a goodbarrier to oxidation, etc.

A typical approach to meeting these requirements is to increase thethickness of the microcapsule wall. But this results in an undesirablereduction in the loading capacity of the microcapsule. That is, the“payload” of the microcapsule, being the mass of the loading substanceencapsulated in the microcapsule divided by the total mass of themicrocapsule, is low. The typical payload of such “single-core”microcapsules made by spray drying an emulsion is in the range of about25-50%.

Another approach to the problem has been to create what are known as“multi-core” microcapsules. These microcapsules are usually formed byspray drying an emulsion of core material such that the shell materialcoats individual particles of core material, which then aggregate andform a cluster. A typical multi-core microcapsule is depicted in priorart FIG. 1. Multi-core microcapsule 10 contains a plurality of cores 12.The cores 12 take the form of entrapped particles of solids or of liquiddroplets dispersed throughout a relatively continuous matrix of shellmaterial 14. As a result, there is a high ratio of shell material toloading material and the payload of the multi-core microcapsule istherefore low. Moreover, despite the high ratio of shell material toloading substance in such microcapsules, the shell material is poorlydistributed. As shown in prior art FIG. 1, many of the cores 12 are veryclose to the surface 16 of the microcapsule. The cores at the surfaceare therefore not well protected against rupture or from oxidation.

Known microcapsules therefore either have a poor payload, or fail toadequately contain and protect the loading substance deposited therein.Moreover, because these microcapsules are generally prepared in a singlestep, it is difficult to incorporate multiple functionalities, such asoxidation resistance, moisture resistance and taste masking into asingle microcapsule.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a multi-core microcapsulecomprising: (a) an agglomeration of primary microcapsules, each primarymicrocapsule comprising a core and a first shell surrounding the core;(b) a second shell surrounding the agglomeration; and (c) a third shellsurrounding the second shell; at least one of the first, second andthird shells comprising a complex coacervate.

In another aspect, the invention provides a single-core microcapsulecomprising: (a) a core; (b) a first shell surrounding the core; and (c)a second shell surrounding the first shell; at least one of the firstand second shells comprising a complex coacervate.

In the case of either the multi-core or single-core microcapsules, it ispreferred that all of the shells comprise a complex coacervate, whichmay be the same or different for each of the shells. Additional shells,e.g. from 1 to 20, may be added to further strengthen the microcapsule.

In another aspect, the invention provides a process for making amicrocapsule having a plurality of shells, the process comprising:

-   (a) providing a microcapsule selected from the group consisting of:    -   (i) a multi-core microcapsule comprising: an agglomeration of        primary microcapsules, each primary microcapsule comprising a        core and a first shell surrounding the core; and a second shell        surrounding said agglomeration; and    -   (ii) a single-core microcapsule comprising: a core; and a first        shell surrounding the core;-   (b) mixing the microcapsule with first and second polymer components    of shell material in aqueous solution;-   (c) adjusting at least one of pH, temperature, concentration and    mixing speed to form shell material comprising the first and second    polymer components, the shell material forming an additional shell    enveloping the microcapsule;    wherein at least one of the first shell, the second shell and the    additional shell comprises a complex coacervate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a typical prior art multi-core microcapsule.

FIGS. 2 and 3 depict embodiments of the invention in which multi-coremicrocapsules are provided having multiple shells.

FIGS. 4 and 5 depict embodiments of the invention in which single-coremicrocapsules are provided having multiple shells.

FIG. 6 is a photomicrograph of multi-core microcapsules prepared with aone-step process (62% payload), prepared for purposes of comparison.

FIG. 7 is a photomicrograph of multi-core microcapsules prepared with atwo-step process in accordance with the invention (59% payload).

FIG. 8 is a photomicrograph of multi-core microcapsules prepared with atwo-step process in accordance with the invention in which alginate isincorporated in the outer shell (53% payload).

FIG. 9 is a photomicrograph of multi-core microcapsules prepared with athree-step process in which lipids and alginate are incorporated in aninner shell while gelatine and polyphosphate forms an outer shell.

FIG. 10 is a photomicrograph of multi-core microcapsules prepared with atwo-step process in which lipids and alginate are incorporated in thesecond shell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Core Materials

Any core material that may be encapsulated in microcapsules is useful inthe invention. Indeed, in certain embodiments, commercially availablemicrocapsules may be obtained and then further processed according tothe processes of the invention.

When the initial multi-core microcapsules are prepared according toprocesses as described herein involving an aqueous solution, the corematerial may be virtually any substance that is not entirely soluble inthe aqueous solution. Preferably, the core is a solid, a hydrophobicliquid, or a mixture of a solid and a hydrophobic liquid. The core ismore preferably a hydrophobic liquid, such as grease, oil or a mixturethereof. Typical oils may be fish oils, vegetable oils, mineral oils,derivatives thereof or mixtures thereof. The loading substance maycomprise a purified or partially purified oily substance such as a fattyacid, a triglyceride or a mixture thereof. Omega-3 fatty acids, such aso-linolenic acid (18:3n3), octadecatetraenoic acid (18:4n3),eicosapentaenoic acid (20:5n3) (EPA) and docosahexaenoic acid (22:6n3)(DHA), and derivatives thereof and mixtures thereof, are preferred. Manytypes of derivatives are well known to one skilled in the art. Examplesof suitable derivatives are esters, such as phytosterol esters, branchedor unbranched C₁-C₃₀ alkyl esters, branched or unbranched C₂-C₃₀ alkenylesters or branched or unbranched C₃-C₃₀ cycloalkyl esters, in particularphytosterol esters and C₁-C₆ alkyl esters. Preferred sources of oils areoils derived from aquatic organisms (e.g. anchovies, capelin, Atlanticcod, Atlantic herring, Atlantic mackerel, Atlantic menhaden, salmonids,sardines, shark, tuna, etc) and plants (e.g. flax, vegetables, algae,etc).

While the core may or may not be a biologically active substance such asa tocopherol, antioxidant or vitamin, the microcapsules of the presentinvention are particularly suited for biologically active substances,for example, drugs, nutritional supplements, flavours, antioxidants ormixtures thereof.

Shell Material

Coacervation is a phase separation phenomenon, in which a homogenouspolymer solution is converted into two phases. One is a polymer-richphase, called a coacervate. The other is a polymer-poor phase, i.e.,solvent. Complex coacervation is caused by the interaction of twooppositely charged polymers.

Preferably, a positively charged polymer component “A” interacts with anegatively charged polymer component “B”. For example, positivelycharged type A gelatine (“component A”) forms complex coacervates withnegatively charged polyphosphate (“component B”). Other systems thathave been studied are gelatine/gum Acacia, gelatine/pectin,gelatine/carboxymethyl guar gum and whey protein/gum arabic.

Component A is preferably gelatine type A, chitosan, etc., althoughother polymers are also contemplated as component A. Component B ispreferably gelatine type B, polyphosphate, gum arabic, alginate,carrageenan, pectin, carboxymethylcellulose, or a mixture thereof.

In addition to the charge density of the two polymer components, complexcoacervation depends on other factors such as molecular weight of thepolymers and their ratio, ionic strength, pH and temperature of themedium (J. Microencapsulation, 2003, Vol. 20, No. 2: 203-210).

The molar ratio of component A:component B that is used depends on thetype of components but is typically from 1:5 to 15:1. For example, whengelatine type A and polyphosphate are used as components A and Brespectively, the molar ratio of component A:component B is preferably8:1 to 12:1; when gelatine type A and gelatine type B are used ascomponents A and B respectively, the molar ratio of componentA:component B is preferably 2:1 to 1:2; and when gelatine type A andalginate are used as components A and B respectively, the molar ratio ofcomponent A:component B is preferably 3:1 to 5:1.

One suitable process of microencapsulation using complex coacervationcomprises three steps: 1) dispersing the loading substance into a systemof at least one of the polymers for the complex coacervate; 2) formingshells by deposition of coacervates which derive from the polymericcomponents under controlled conditions of temperature, pH, concentrationof colloids, mixing speed etc.; and 3) hardening of the shells bycrosslinking of the coacervates deposited on microcapsules (Ullmann'sEncyclopedia of Industrial Chemistry 6^(th) edition. 2001, Vol. A16. pp.575-588).

Any shells that do not comprise complex coacervates may be formed of anymaterial that can form an additional shell around the microcapsule. Theadditional shell material typically comprises at least one polymercomponent. Examples of polymer components include, but are not limitedto, proteins, e.g. gelatines, soy proteins, whey proteins, and milkproteins, polyphosphate, polysaccharides and mixtures thereof. Preferredpolymer components are gelatine A, gelatine B, polyphosphate, gumarabic, alginate, chitosan, carrageenan, pectin, cellulose orderivatives of cellulose such as carboxymethylcellulose (CMC) or amixture thereof. A particularly preferred form of gelatine type A has aBloom strength of 50-350, more preferably a Bloom strength of about 275.

The shell material can also comprise lipids, such as waxes, fatty acidsand oils, etc. to provide desired functionalities. The incorporation oflipids into the shell material improves the impermeability of the shellto water and oxygen. A preferred lipid for this purpose is beeswax.These lipids may be in solid, semi-solid or liquid form.

Processing Aids

Processing aids may be included in the shell material. Processing aidsmay be used for a variety of reasons. For example, they may be used topromote agglomeration of primary microcapsules when forming multi-coremicrocapsules, control microcapsule size and shape and/or to act as anantioxidant. Antioxidant properties are useful both during the process(e.g. during coacervation and/or spray drying) and in the microcapsulesafter they are formed (e.g. to extend shelf-life of loading substanceswhich are readily oxidized, etc). Preferably a small number ofprocessing aids that perform a large number of functions are used. Forexample, ascorbic acid or a salt thereof may be used to promoteagglomeration of the primary microcapsules, to control microcapsule sizeand shape and to act as an antioxidant. The ascorbic acid or saltthereof is preferably used in an amount of about 100 ppm to about 10,000ppm, more preferably about 1000 ppm to about 5000 ppm relative to thebatch size (i.e., the total weight). A salt of ascorbic acid, such assodium or potassium ascorbate, is particularly preferred in thiscapacity. Other processing aids include, without limitation, bufferingacids and/or their salts such as phosphoric acid, acetic acid, citricacid, and the like.

Structure of Microcapsules

In one embodiment, microcapsules of the invention have a structuregenerally as depicted in FIG. 2. FIG. 2 depicts a multi-coremicrocapsule prepared according to a multi-step process of theinvention. Primary microcapsules comprise cores 18 (i.e. the loadingsubstance) surrounded by first shells 20. The primary microcapsulesagglomerate and the space 22 between them is usually at least partlyfilled by additional shell material of same composition as first shell20, although there may be voids between some of the primarymicrocapsules. The agglomeration of primary microcapsules is surroundedby a second shell 24.

Multi-core microcapsules comprising second shell 24 may be preparedaccording to the processes described herein and exemplified in theexamples or by generally the same techniques that are described inApplicant's co-pending U.S. patent application Ser. No. 10/120,621 filedApr. 11, 2002, corresponding to International Application No.PCT/CA2003/000520 filed Apr. 8, 2003, the disclosures of both of whichare incorporated herein by reference. These multi-core microcapsules areparticularly useful because the foam-like structure of primarymicrocapsules, supported by additional shell material in space 22 andsurrounded by second shell 24 is an extremely strong, rupture-resistantstructure that has a high payload i.e. the ratio of the total mass ofthe cores to the total mass of the multi-core microcapsule is very high,e.g. at least 50, 55, 60, 65, 70, 75, 80, 85, 90% or higher. This iscalled a “one-step” process when shells 20 and 24 are of the samecomposition and formed in a single step. When shells 20 and 24 are ofdifferent composition, the process involves two steps.

Commercially available multicore microcapsules may also be used asstarting materials. An example is the Driphorm™ Hi-DHA™microencapsulated tuna oil, manufactured by Nu-Mega Ingredients Pty.Ltd., Queensland, AU.

In accordance with the invention, a three-step process takes place whena third shell 26 is formed on the multi-core microcapsule. Third shell26 further strengthens the microcapsule and can be advantageously usedto provide a shell having properties different from those of shell 24.

For instance, different polymer components can be incorporated intothird shell 26. In addition, or alternatively, lipids may beincorporated into shell 26 to increase moisture or oxygen impermeabilityor the like. These properties might instead be incorporated into secondshell 24 rather than third shell 26 (or also into second shell 24 aswell as into third shell 26), depending on the requirements for aparticular purpose. Additional shells, not shown in FIG. 2, may beformed around third shell 26, by the methods and techniques of theinvention. For instance, N additional shells could be added, wherein Nis an integer from 1 to 20.

At least one of shells 20, 24 and 26 and of any additional shellscomprises a complex coacervate, as described above. Preferably, at leasttwo of the shells comprise a complex coacervate. Even more preferably,all of the shells comprise a complex coacervate. For instance, thefollowing shells may comprise complex coacervates: (a) shell 20; (b)shell 24; (c) shell 26; (d) shells 20 and 24; (e) shells 20 and 26; (f)shells 24 and 26; or (g) shells 20, 24 and 26. Additional shells alsopreferably comprise a complex coacervate.

Referring again to FIG. 2, the primary microcapsules (i.e. cores 18surrounded by first shells 20) typically have an average diameter ofabout 40 nm to about 10 μm, more particularly from about 0.1 μm to about5 μm, even more particularly an average diameter of about 1-2 μm. Thefinished multi-core microcapsule, i.e. including third shell 26, usuallyhas an average diameter from about 1 μm to about 2000 μm, more typicallyfrom about 20 μm to about 1000 μm, more particularly from about 20 μm toabout 100 μm and even more particularly from about 50 μm to about 100μm.

In FIG. 2, second shell 24 and third shell 26 are depicted as discretelayers. This will be the case if the shells are formed of the differentshell materials. In that case, even if they do not differ in appearance,they will have a different composition and can be represented asdiscrete, distinct layers. But if second shell 24 and third shell 26 areformed of the same shell material, they may, as shown in FIG. 3, mergeto form a single, continuous layer, having the combined thickness ofsecond shell 24 and third shell 26. As shown in FIG. 3, when the secondand third shells are of the same composition, there may be no discreteboundary separating them. This would be true also in microcapsules ofthe invention having fourth or additional shells that are of the samecomposition as the preceding shell.

The invention is also useful in the preparation of single-coremicrocapsules having multiple shells. Single-core microcapsules usefulas starting materials are commercially available. Examples includemicroencapsulated flavours by Givaudan Flavors Corp., Cincinnati, Ohio,USA, and microencapsulated minerals and vitamins by Watson Food Co.Inc., West Haven, Conn., USA. Alternatively, they can be made by complexcoacervation processes as described herein, e.g. by preparing primarymicrocapsules without a further agglomeration step. FIG. 4 depicts asingle-core microcapsule having multiple shells in accordance with theinvention. Core 18 is surrounded by a first shell 20 and a second shell24. Additional shells, not shown in FIG. 4, may be formed around secondshell 24, by the methods and techniques of the invention. For instance,N additional shells could be added, wherein N is an integer from 1 to20.

As with the multi-core microcapsules, shells 20 and 24 of single-coremicrocapsules may be of the same or different composition. At least oneof shells 20 and 24 and of any additional shells comprises complexcoacervates as described above. Preferably, at least two of the shellscomprise a complex coacervate. Even more preferably all of the shellscomprise a complex coacervate. For instance, the following shells maycomprise complex coacervates: (a) shell 20; (b) shell 24; or (c) shells20 and 24. Additional shells also preferably comprise complexcoacervates.

Single-core microcapsules may be as large as multi-core microcapsules.For instance, the exterior diameter of second shell 24 in thesingle-core microcapsule of FIG. 4 may be from about 1 μm to about 2000μm. More typically it will be from about 20 μm to about 1000 μm, moreparticularly from about 20 μm to about 100 μm and even more particularlyfrom about 50 μm to about 100 μm.

When they are of the same composition, first shell 20 and second shell24 (and any additional shell) of the single-core multicapsule may mergeto form a single continuous layer as depicted in FIG. 5. This may bedone in a one-step process.

Processes

Single or multi-core microcapsules to which additional shells may beadded by the processes of the invention may be obtained from commercialsources. In a particularly preferred embodiment, multi-coremicrocapsules prepared in accordance with applicant's co-pending U.S.patent application Ser. No. 10/120,621 filed Apr. 11, 2002,corresponding to International Application No. PCT/CA2003/000520 filedApr. 8, 2003, the disclosures of both of which are incorporated hereinby reference, are used. Such microcapsules can be prepared e.g. by a onestep process as follows.

An aqueous mixture of a loading substance (i.e.

core material) and a polymer component of the shell material is formed.The aqueous mixture may be a mechanical mixture, a suspension or anemulsion. When a liquid loading material is used, particularly ahydrophobic liquid, the aqueous mixture is preferably an emulsion of theloading material and the polymer components.

In a more preferred aspect, a first polymer component is provided inaqueous solution, preferably together with processing aids, such asantioxidants. A loading substance may then be dispersed into the aqueousmixture, for example, by using a homogenizer. If the loading substanceis a hydrophobic liquid, an emulsion is formed in which a fraction ofthe first polymer component begins to deposit around individual dropletsof loading substance to begin the formation of primary shells. If theloading substance is a solid particle, a suspension is formed in which afraction of the first polymer component begins to deposit aroundindividual particles to begin the formation of primary shells. At thispoint, another aqueous solution of a second polymer component may beadded to the aqueous mixture.

Droplets or particles of the loading substance in the aqueous mixturepreferably have an average diameter of less than 100 μm, more preferablyless than 50 μm, even more preferably less than 25 μm. Droplets orparticles of the loading substance having an average diameter less than10 μm or less than 5 μm or less than 3 μm or less than 1 μm may be used.Particle size may be measured using any typical equipment known in theart, for example, a Coulter™ LS230 Particle Size Analyzer, Miami, Fla.,USA.

The amount of the polymer components of the shell material provided inthe aqueous mixture is typically sufficient to form both the primary andouter shells of microcapsules. Preferably, the loading substance isprovided in an amount of from about 1% to about 15% by weight of theaqueous mixture, more preferably from about 3% to about 8% by weight,and even more preferably about 6% by weight.

If a complex coacervate is desired, the pH, temperature, concentration,mixing speed or a combination thereof is then adjusted to accelerate theformation of the primary shells of complex coacervate around thedroplets or particles of the loading substance to form primarymicrocapsules. In the case of multicore microcapsules, agglomeration ofthe primary microcapsules will take place to form discrete clumps atdesired size and shape.

pH is an expression of the concentration of hydrogen ions in solution.Such ions affect the ionization equilibria of the component A and Bpolymers involved in complex coacervation and thus the formation ofcomplex coacervates. The pH is adjusted so that the component A polymerwill bear a net positive charge and the component B polymer will bear anet negative charge. Hence, the pH adjustment depends on the type ofshell material to be used.

For example, when gelatine type A is a polymer component, the gelatinemolecules have nearly equal positive and negative charges (i.e. zero netpolarity change) at their point of zero charge (pzc) around pH 9-10.Only when the solution pH is lower than the pzc value, will the polymerbear a net positive charge, which interacts with the negatively chargedcomponent B (e.g. gum arabic, polyphosphate, alginate, etc.).

In the case of gelatine type A, the pH is preferably adjusted to a valuefrom 3.5-5.0, more preferably from 4.0-5.0. Much outside this range, thegelatine-based complex tends to form gels upon cooling rather than ashell on the microcapsules. If the pH of the mixture starts in thedesired range, then little or no pH adjustment is required.

The molar ratio of components A and B is adjusted to favour formation ofshells on the microcapsules rather than merely the formation of gelparticles in solution. Suitable molar ratios are discussed above underthe heading “Shell Material”.

The concentration of components A and B in the aqueous mixture may alsoaffect the formation of complex coacervates and can be adjustedaccordingly. Typically, the total concentration of components A and Bvaries from 1% to 20%, preferably 2-10%, and more preferably 3-6% byweight of the aqueous mixture. For instance, when gelatine type A isused as component A, the concentration of gelatine type A is preferablyfrom 1-15% by weight of the aqueous mixture, more preferably 2-6% byweight and even more preferably 2-4% by weight. Similarly, whenpolyphosphate is used as component B, its concentration in the aqueousmixture is preferably 0.01-0.65% by weight of the aqueous mixture, morepreferably 0.13-0.17% by weight, even more preferably 0.13-0.26% byweight.

The initial temperature of the aqueous mixture is preferably set to avalue of from about 40° C. to about 60° C., more preferably at about 50°C.

Mixing speed influences the deposition of complex coacervates on thesurface of microcapsules. If the mixing speed is too low, the aqueousmixture is agitated insufficiently and undesirably large microcapsulesmay be formed. Conversely, if the mixing speed is too high, high shearforces are generated and prevent shell material from forming on themicrocapsules. Instead, gel particles form in the solution. The mixingspeed is preferably between 100 and 1500 rpm, more preferably between400 and 1000 rpm and even more preferably between 600 and 800 rpm.Particular mixing parameters depend on the type of equipment being used.Any of a variety of types of mixing equipment known in the art may beused. Particularly useful is an axial flow impeller, such as Lightnin™A310 or A510.

At this time, materials for outer shell are added into the mixture, andthe aqueous mixture may then be cooled under controlled cooling rate andmixing parameters to permit coating of the primary microcapsules to formouter shells. It is advantageous to control the formation of the outershell at a temperature above the gel point of the shell material. It isalso possible at this stage to further add more polymer components,either of the same kind or a different kind, in order to thicken theouter shell and/or produce microcapsules having different layers ofshells to provide desired functionalities. The temperature is preferablylowered at a rate of about 1° C./10 minutes until it reaches atemperature of from about 5° C. to about 10° C., preferably about 5° C.The outer shell encapsulates the primary microcapsules or clumps to forma rigid encapsulated agglomeration of microcapsules.

At this stage, a cross-linker may be added to further increase therigidity of the microcapsules by cross-linking the shell material inboth the outer and primary shells and to make the shells insoluble inboth aqueous and non-aqueous (e.g., oil) media. Any suitablecross-linker may be used and the choice of cross-linker depends somewhaton the choice of shell material. Preferred cross-linkers are enzymaticcross-linkers (e.g. transglutaminase), aldehydes (e.g. formaldehyde orgluteraldehyde), tannic acid, alum, organic or inorganic calcium orpotassium salt, or a mixture thereof. When the microcapsules are to beused to deliver a biologically active substance to an organism, thecross-linkers are preferably non-toxic or of sufficiently low toxicity.The type and the amount of cross-linker used depend on the type of shellmaterial and may be adjusted to provide more or less structural rigidityas desired. For example, when gelatine type A is used in the shellmaterial, transglutaminase may be conveniently used in an amount ofabout 0.2% to about 2.0%, preferably about 1.0%, by weight ofmicrocapsule suspension. In general, one skilled in the art mayroutinely determine the desired amount in any given case by simpleexperimentation.

At this stage, multi-core microcapsules have been produced. Thesemicrocapsules or other microcapsules may then be processed in accordancewith the invention to add additional shell layers as described above.Preferably, additional shells are added after the formation of the outershell of the microcapsule or before the cross-linking step. Moreparticularly, first and second polymer components of shell material aredissolved in aqueous solution e.g. at 40 to 60° C., more preferablyaround 50° C. pH may be controlled or adjusted at this stage. Themicrocapsules previously prepared are then combined with this mixture.Alternatively, the microcapsules may be combined with an aqueoussolution of the first polymer component of shell material and then asecond aqueous solution of the second polymer component of shellmaterial may be added. pH, temperature, concentration, mixing speed or acombination thereof can then be adjusted as described above so that thepolymer components of shell material form a complex coacervatesurrounding and coating the microcapsules with an additional shell. Asdiscussed above, processing aids may be incorporated as may behydrophobic materials such as oils, waxes, resins or fats. The new outershell may be then cross-linked as described above. These additionalsteps of forming additional shell layers may be repeated as desired tobuild up a suitable number of further shells on the microcapsule.

Finally, the microcapsules may be washed with water and/or dried toprovide a free-flowing powder. Drying may be accomplished by a number ofmethods known in the art, such as freeze drying, drying with ethanol orspray drying. Spray drying is a particularly preferred method for dryingthe microcapsules. Spray drying techniques are disclosed in “SprayDrying Handbook”, K. Masters, 5^(th) edition, Longman ScientificTechnical UK, 1991, the disclosure of which is hereby incorporated byreference.

Uses

The microcapsules produced by the processes of the present invention maybe used to prepare liquids as free-flowing powders or compressed solids,to store a substance, to separate reactive substances, to reducetoxicity of a substance, to protect a substance against oxidation, todeliver a substance to a specified environment and/or to control therate of release of a substance. In particular, the microcapsules may beused to deliver a biologically active substance to an organism fornutritional or medical purposes. The biologically active substance maybe, for example, a nutritional supplement, a flavour, a drug and/or anenzyme. The organism is preferably a mammal, more preferably a human.Microcapsules containing the biologically active substance may beincluded, for example, in foods or beverages or in drug deliverysystems. Use of the microcapsules of the present invention forformulating a nutritional supplement into human food is particularlypreferred.

Microcapsules of the present invention have good rupture strength tohelp reduce or prevent breaking of the microcapsules duringincorporation into food or other formulations. Furthermore, themicrocapsules' shells can be formulated to be insoluble in both aqueousand non-aqueous (e.g., oil) media, and help reduce or prevent oxidationand/or deterioration of the loading substance during preparation of themicrocapsules, during long-term storage, and/or during incorporation ofthe microcapsules into a formulation vehicle, for example, into foods,beverages, nutraceutical formulations or pharmaceutical formulations.

The invention will now be further illustrated by the followingnon-limiting examples.

Examples Example 1 Multicore Microcapsules Prepared by One-Step Processfor Comparison (both First and Second Shells having the Same Compositionof Gelatine and Polyphosphate)

54.5 grams gelatine 275 Bloom type A (isoelectric point of about 9) wasmixed with 600 grams of deionized water containing 0.5% sodium ascorbateunder agitation at 50° C. until completely dissolved. 5.45 grams ofsodium polyphosphate was dissolved in 104 grams of deionized watercontaining 0.5% sodium ascorbate. 90 grams of a fish oil concentratecontaining 30% eicosapentaenoic acid ethyl ester (EPA) and 20%docosahexaenoic acid ethyl ester (DHA) (available from Ocean NutritionCanada Ltd.) was dispersed with 1.0% of an antioxidant (mixed naturaltocopherols) into the gelatine solution with a high speed Polytron™homogenizer at 5,500 rpm for 6 minutes. An oil-in-water emulsion wasformed. The oil droplet size had a narrow distribution with an averagesize of about 1 μm measured by Coulter™ LS230 Particle Size Analyzer.The emulsion was diluted with 700 grams of deionized water containing0.5% sodium ascorbate at 50° C. The sodium polyphosphate solution wasthen added into the emulsion and mixed with a Lightnin™ agitator at 600rpm. The pH was then adjusted to 4.5 with a 10% aqueous acetic acidsolution. During pH adjustment and the cooling step that followed pHadjustment, a coacervate formed from the gelatine and polyphosphatecoated onto the oil droplets to form primary microcapsules. Cooling wascarried out to above the gel point of the gelatine and polyphosphate andthe primary microcapsules started to agglomerate to form lumps underagitation. Upon further cooling of the mixture, polymer remaining in theaqueous phase further coated the lumps of primary microcapsules to forman encapsulated agglomeration of microcapsules having an outer shell andhaving an average size of 50 μm. Once the temperature had been cooled to5° C., 2.7 grams of 50% gluteraldehyde was added into the mixture tofurther strengthen the shell. The mixture was then warmed to roomtemperature and kept stirring for 12 hours. Finally, the microcapsulesuspension was washed with water. The washed suspension was then spraydried to obtain a free-flowing powder. A payload of 62% was obtained.

Example 2

A Two-Step Process with Gelatine and Polyphosphate in both First andSecond Shells, but having Different Compositions

Step A: 15.6 grams gelatine 275 Bloom type A (isoelectric point of about9) was mixed with 172 grams of deionized water containing 0.5% sodiumascorbate under agitation at 50° C. until completely dissolved. 1.56grams of sodium polyphosphate was dissolved in 29.7 grams of deionizedwater containing 0.5% sodium ascorbate. 69 grams of a fish oilconcentrate containing 30% eicosapentaenoic acid ethyl ester (EPA) and20% docosahexaenoic acid ethyl ester (DHA) (available from OceanNutrition Canada Ltd.) was dispersed with 1.0% of an antioxidant (mixednatural tocopherols) into the gelatine solution with a high speedPolytron™ homogenizer at 6,100 rpm for 4 minutes. An oil-in-wateremulsion was formed. The oil droplet size had a narrow distribution withan average size of about 1 μm measured by Coulter™ LS230 Particle SizeAnalyzer. The emulsion was diluted with 319 grams of deionized watercontaining 0.5% sodium ascorbate at 50° C. The sodium polyphosphatesolution was then added into the emulsion and mixed with a Lightnin™agitator at 600 rpm. The pH was then adjusted to 4.5 with a 10% aqueousphosphoric acid solution. During pH adjustment and the cooling step thatfollowed pH adjustment, a coacervate formed from the gelatine andpolyphosphate coated onto the oil droplets to form primarymicrocapsules, and then the primary microcapsules started to agglomerateto form lumps under agitation. A payload of 80% was obtained at thisstep.

Step B: A gelatine solution was prepared by dissolving 41.8 grams ofgelatine 275 Bloom type A (isoelectric point of about 9) in 460 grams ofdeionized water containing 0.5% sodium ascorbate under agitation at 50°C. until completely dissolved. A sodium polyphosphate solution wasprepared by dissolving 4.18 grams of sodium polyphosphate in 79.5 gramsof deionized water containing 0.5% sodium ascorbate. The gelatine andpolyphosphate solutions were combined to form a mixture, and pH of themixture was adjusted to 4.7 with 10% aqueous phosphoric acid.

Step C: The mixture from Step B was added to the mixture with lumpsformed in step A. Cooling was carried out under agitation to cause thegelatine and polyphosphate to form coacervates and to coat the lumpsformed in Step A to form an outer shell. The microcapsules thus formedhad an average size of 60 μm. Once the temperature had been cooled to 5°C., 2.1 grams of 50% gluteraldehyde was added into the mixture tofurther strengthen the shell. The mixture was then warmed to roomtemperature and stirred continuously for 12 hours. Finally, themicrocapsule suspension was washed with water. The washed suspension wasthen spray dried to obtain a free-flowing powder. A payload of 59% wasobtained.

Example 3 A Two-Step Process having Gelatine and Alginate in the SecondShell

Step A: Same as Step A in Example 2.

Step B: A gelatine solution was prepared by dissolving 23.0 grams ofgelatine 275 Bloom type A (isoelectric point of about 9) in 371 grams ofdeionized water under' agitation at 50° C. until completely dissolved. Asodium alginate (ISP Alginates) solution was prepared by dissolving 3.00grams of sodium alginate in 503.8 grams of deionized water. The gelatineand sodium alginate solutions were combined to form a mixture. The pH ofthe mixture was adjusted to 5.00 with 10% aqueous phosphoric acid.

Step C: The mixture from Step B was added to the mixture with lumpsformed in step A. Cooling was carried out under agitation to causegelatine and alginate to form coacervates and coat the lumps formed inStep A to form an outer shell. The microcapsules thus formed had anaverage size of around 80 μm. Once the temperature had been cooled to 5°C., 2.1 grams of 50% gluteraldehyde was added into the mixture tofurther strengthen the shell. The mixture was then warmed to roomtemperature and stirred continuously for 12 hours. Finally, themicrocapsule suspension was washed with water. The washed suspension wasthen spray dried to obtain a free-flowing powder. A payload of 53% wasobtained.

Example 4 A Three-Step Process to Incorporate Wax and Alginate in theSecond Shell and Alginate in the Third Shell

Step A: 20.0 grams gelatine 275 Bloom type A (isoelectric point of about9) was mixed with 220.1 grams of deionized water containing 0.5% sodiumascorbate under agitation at 50° C. until completely dissolved. 2.00grams of sodium polyphosphate was dissolved in 38.0 grams of deionizedwater. 88.0 grams of a fish oil concentrate containing 30%eicosapentaenoic acid ethyl ester (EPA) and 20% docosahexaenoic acidethyl ester (DHA) (available from Ocean Nutrition Canada Ltd.) wasdispersed with 1.0% of an antioxidant (mixed natural tocopherols) intothe gelatine solution with a high speed Polytron™ homogenizer at 6,100rpm for 4 minutes. An oil-in-water emulsion was formed. The oil dropletsize had a narrow distribution with an average size of about 1 μmmeasured by Coulter™ LS230 Particle Size Analyzer. The emulsion wasdiluted with 408.6 grams of deionized water at 50° C. The sodiumpolyphosphate solution was then added into the emulsion and mixed with aLightnin™ agitator at 600 rpm. The pH was then adjusted to 4.5 with a10% aqueous phosphoric acid solution. During pH adjustment and thecooling step that followed pH adjustment, a coacervate formed from thegelatine and polyphosphate coated onto the oil droplets to form primarymicrocapsules, and then the primary microcapsules started to agglomerateto form lumps under agitation. A payload of 80% was obtained at thisstep.

Step B: A gelatine solution was prepared by dissolving 8.6 grams ofgelatine 275 Bloom type A (isoelectric point of about 9) in 94.5 gramsof deionized water under agitation at 65° C. until completely dissolved.25.8 grams of beeswax melted at 65° C. was emulsified in the gelatinesolution with a high speed Polytron™ homogenizer at 6,100 rpm for 4minutes. A wax-in-water emulsion was formed. An alginate solution wasprepared by dissolving 2.3 grams of sodium alginate in 192 grams ofdeionized water was added to the emulsion, and pH of the mixture wasadjusted to 4.7 with 10% aqueous phosphoric acid. The mixture was thenadded into lump mixtures in step A under agitation at 800 rpm, andcooling was carried out to cause the gelatine-alginate-wax compositematerial to form a coating onto the lumps formed in Step A to formmicrocapsules. A payload of 60% was obtained at this step.

Step C: A solution was prepared by dissolving 23.1 grams of gelatine and2.3 grams of sodium alginate in 384.9 grams of deionized water underagitation at 50° C. until completely dissolved. pH of the mixture wasadjusted to 4.5 with 10% aqueous phosphoric acid, and the mixture wasthen added into microcapsule mixtures formed in step B under agitationat 800 rpm. Cooling was carried out to cause the gelatine-alginatematerial to form a coating onto the microcapsules that formed in Step B.Once the temperature had been cooled to 5° C., 1.5 grams oftransglutaminase was added into the mixture to cross-link the shell. Themixture was then warmed to room temperature and kept stirring for 12hours. Finally, the microcapsule suspension was spray dried to obtain afree-flowing powder. A final payload of 52% was obtained.

Example 5 A Two-Step Process of Multicore Microcapsules having Wax andAlginate in the Second Shell

Step A: 13.0 grams of gelatine 275 Bloom type A (isoelectric point ofabout 9) was mixed with 143.0 grams of deionized water containing 0.5%sodium ascorbate under agitation at 50° C. until completely dissolved.1.3 grams of sodium polyphosphate was dissolved in 24.7 grams ofdeionized water. 57.2 grams of fish oil containing 18% eicosapentaenoicacid (EPA) and 12% docosahexaenoic acid (DHA) (available from OceanNutrition Canada Ltd.) was dispersed with 1.0% of an antioxidant (mixednatural tocopherols) into the gelatine solution with a high speedPolytron™ homogenizer at 8,000 rpm for 4 minutes. An oil-in-wateremulsion was formed. The oil droplet size had a narrow distribution withan average size of about 1 μm measured by Coulter™ LS230 Particle SizeAnalyzer. The emulsion was diluted with 266.0 grams of deionized waterat 50° C. The sodium polyphosphate solution was then added into theemulsion and mixed with a Lightnin™ agitator at 350 rpm. The pH was thenadjusted to 4.4 with a 10% aqueous phosphoric acid solution. During pHadjustment and the cooling step that followed pH adjustment, acoacervate formed from the gelatine and polyphosphate coated onto theoil droplets to form primary microcapsules, and then the primarymicrocapsules started to agglomerate to form lumps under agitation. Apayload of 80% was obtained at this step.

Step B: A gelatine solution was prepared by dissolving 7.05 grams ofgelatine 275 Bloom type A (isoelectric point of about 9) in 77.9 gramsof deionized water under agitation at 70° C. until completely dissolved.7.05 grams of beeswax melted at 70° C. was emulsified in the gelatinesolution with a high speed Polytron™ homogenizer at 8,000 rpm for 4minutes. A wax-in-water emulsion was formed. An alginate solution (45 °C.) was prepared by dissolving 7.62 grams of sodium alginate in 630grams of deionized water was added to the emulsion, and pH of themixture was adjusted to 5.3 with 10% aqueous phosphoric acid. Themixture was then added into lump mixtures in step A under agitation at450 rpm followed by adjusting the pH value of the mixture to 4.9, andcooling was carried out to cause the gelatine-alginate-wax compositematerial to form a coating onto the lumps formed in Step A to formmicrocapsules. Once the temperature had been lowered to 5° C., 3.8 gramsof transglutaminase was added into the mixture to cross-link the shells.The mixture was then warmed up to room temperature and stirred at 600rpm for 12 hours. Finally, the microcapsule suspension was spray driedto obtain a free-flowing powder. A final payload of 57% was obtained.

Example 6 Evaluation of Microcapsules

Images of microcapsules of Examples 1-5 are shown in FIG. 6 to FIG. 10,respectively. It can be seen clearly that at approximately the samepayload (60%) the microcapsules prepared with a two step process (FIG.7) have much thicker outer shells than those prepared with one stepprocess (FIG. 6). The microcapsules prepared with a three step processhaving a composite shell containing lipids (FIG. 9) clearly show thelipid droplets incorporated in the second shell and near theagglomerated oil core.

Accelerated oxidative stability in dry state was evaluated by placingthe prepared microcapsule powders from each of Examples 1-4 in an oxygenbomb (Oxipres™, MIKROLAB AARHUS A/S, Denmark) with an initial oxygenpressure of 5 bar at a constant temperature of 65° C. When theencapsulated fish oil started to oxidize, the oxygen pressure dropped,and an induction period or time was determined. A longer inductionperiod means that the contents of the microcapsules are better protectedtowards oxidation.

Induction periods are shown in Table 1. The microcapsules made from atwo-step process in accordance with the invention have higher inductionperiod (50-56 hours) than those made from a one-step process (41 hours).This translates to 22.0% to 37.6% increase in oxidative stability.

TABLE I Comparison of the microcapsules described in Examples 1-5.Induction Loading period Example # Figure # Description (%) (hr) 1 6Multicore one-step 62 41 process for comparison 2 7 Two-step process 5950 with gelatine and polyphosphate in outer shell 3 8 Two-step process53 55 with alginate in outer shell 4 9 Three-step process 52 44incorporating wax and alginate in the second shell and gelatine andpolyphosphate in the third shell 5 10 Two-step process 57 56incorporating wax and alginate in the shell

All publications cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference. The citation ofany publication should not be construed as an admission that suchpublication is prior art.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this specification that certain changesor modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A multi-core microcapsule comprising: (a) an agglomeration of primarymicrocapsules, each primary microcapsule comprising a core and a firstshell surrounding said core; (b) a second shell surrounding saidagglomeration; and (c) a third shell surrounding said second shell; atleast one of said first, second and third shells comprising a complexcoacervate.
 2. The multi-core microcapsule according to claim 1, whereineach of said first, second and third shells comprises a complexcoacervate.
 3. The multi-core microcapsule according to claim 1, whereineach of said first, second and third shells comprises the same complexcoacervate.
 4. The multi-core microcapsule according to claim 1, whereinat least one of said first, second and third shells comprises a complexcoacervate that is different than a complex coacervate that forms one ofthe other shells.
 5. The multi-core microcapsule according to claim 1,wherein said complex coacervate comprises at least one polymer componentselected from the group consisting of: a protein, a polyphosphate, apolysaccharide, gum arabic, alginate, chitosan, carrageenan, pectin,cellulose and cellulose derivatives.
 6. The multi-core microcapsuleaccording to claim 5, wherein said protein is selected from the groupconsisting of gelatine type A, gelatine type B, soy proteins, wheyproteins, milk proteins, and combinations thereof.
 7. The multi-coremicrocapsule according to claim 1, wherein at least one of said first,second and third shells comprises a complex coacervate between gelatineA and at least one polymer component selected from the group consistingof gelatine type B, polyphosphate, gum arabic, alginate, chitosan,carrageenan, pectin and carboxymethylcellulose.
 8. The multi-coremicrocapsule according to claim 1, wherein at least one of said first,second and third shells is a complex coacervate between gelatine A andpolyphosphate.
 9. The multi-core microcapsule according to claim 1,further comprising at least one additional shell surrounding said thirdshell.
 10. The multi-core microcapsule according to claim 9, whereinsaid at least one additional shell surrounding said third shellcomprises a complex coacervate.
 11. The multi-core microcapsuleaccording to claim 1, wherein at least one of said first second andthird shells comprises an antioxidant.
 12. The multi-core microcapsuleaccording to claim 1, wherein at least one of said first, second andthird shells comprises one or more hydrophobic components selected fromthe group consisting of waxes, oils, resins, and fats.
 13. Themulti-core microcapsule according to claim 1, wherein at least one ofsaid first, second and third shells comprises a complex coacervate thatis cross-linked with a cross-linker.
 14. The multi-core microcapsuleaccording to claim 1, wherein said cores comprise at least 50% of thetotal mass of the multi-core microcapsule.
 15. The multi-coremicrocapsule according to claim 1, having an exterior average diameterof from about 1 μm to about 2000 μm, and wherein said first shells havean average diameter of from about 40 nm to about 10 μm. 16-45.(canceled)